Gene activation involves the ordered recruitment of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and subsequent transcript elongation. These events must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent the chromatin block, eukaryotic cells possess a set of chromatin remodeling and nucleosome modifying complexes. The former (e.g. the SWI/SNF complex) use ATP to drive conformational changes in nucleosomes and to move nucleosomes along DNA; the latter contain enzymatic activities (e.g., histone acetylases) which modify the histones post-translationally to mark them for recognition by other complexes. Geneticists have described many interesting connections between chromatin components and transcription, but a system to investigate the structural basis of these has been lacking. We have developed such a model system, involving native plasmid chromatin purified from the yeast Saccharomyces cerevisiae, to perform high resolution studies of the chromatin structures of active and inactive genes. Remarkably, they reveal that activation correlates with large scale movements of nucleosomes and conformational changes within nucleosomes over entire genes. Remodeling of CUP1 chromatin and targeted histone acetylation We chose budding yeast as a model organism because biochemical studies of chromatin structure could be combined with molecular genetics. The CUP1 gene was chosen because its regulation is well understood: CUP1 encodes a metallothionein required to protect cells from the toxic effects of copper in the medium. Copper ions enter the cell and bind to the N-terminal domain of a transcriptional activator, Ace1p, resulting in the folding of this protein to form a DNA-binding domain that recognizes upstream activating sequences in the CUP1 promoter. The changes occurring in the chromatin structure of CUP1 on activation were determined by mapping nucleosome positions at high resolution using the monomer extension method. This revealed that induction results in the Ace1p-dependent movement (sliding) of nucleosomes over the entire CUP1 gene, not just at the promoter. Transcription is not responsible for this movement because the TATA boxes are not required. We propose that a chromatin remodeling activity capable of sliding nucleosomes along CUP1 DNA is recruited by Ace1p to CUP1. Our observations provide direct evidence in vivo for nucleosome movement as a result of CUP1 induction and indicate that remodeling is not restricted to the promoter, but occurs over a chromatin domain defined by CUP1 and its flanking sequences. DNA sequence plays a major role in determining nucleosome positions on CUP1 in vivo: it contains information specifying many overlapping positions which is apparently used by remodeling machines when moving nucleosomes around on CUP1 in vivo. Induction of CUP1 results in targeted acetylation of both histones H3 and H4 at the CUP1 promoter. Targeted acetylation requires Ace1p and the TATA boxes, suggesting that acetylation occurs when TBP binds to the TATA boxes or at a later stage in initiation. Since current models suggest that the function of targeted acetylation is to facilitate the binding of TBP to TATA boxes in nucleosomes, this was surprising and is a major focus of our current work. The histone acetyltransferase (HAT) activity responsible for targeted acetylation at CUP1 was identified by testing various HAT mutants for growth defects in copper. We found that disruption of the putative HAT encoded by the SPT10 gene is lethal at high copper concentrations, and correlates with impaired CUP1 induction and loss of targeted acetylation. SPT10 has been identified by others as a global regulator of core promoter activity, which fits nicely with the TATA box requirement for acetylation at CUP1. We suggest that acetylation might facilitate the binding of Ace1p to nucleosomal binding sites. Our current work has the following aims: (1) Identification of the remodeling complex responsible for nucleosome sliding on CUP1. (2) Determination of the order of recruitment of factors at the CUP1 promoter after copper-induction using chromatin immunoprecipitation (ChIP) experiments. (3) Elucidation of the role of the TATA boxes in targeted acetylation of nucleosomes at the CUP1 promoter. The SWI/SNF Complex and Remodeling of HIS3 Current models for the role of the SWI/SNF ATP-dependent chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur. However, using our plasmid model system with HIS3, a SWI/SNF-regulated gene, we discovered that induction of HIS3 creates a domain of remodeled chromatin structure that extends far beyond the promoter, to include the entire gene. Induction results in a dramatic loss of nucleosomal supercoiling, a decompaction of the chromatin, and a general increase in the accessibility of the chromatin to restriction enzymes. Formation of this domain requires the SWI/SNF complex and the activator Gcn4p, but not the TATA boxes, indicating that remodeling is not the result of transcription. The implication is that the nucleosomes have been opened up in a SWI/SNF-dependent remodeling reaction. We propose that the SWI/SNF complex is recruited to the HIS3 promoter by Gcn4p and then directs remodeling of a chromatin domain. This might facilitate transcription through nucleosomes, in keeping with a possible role for the SWI/SNF complex as an elongation factor. Our current work has the following aims: (1) Elucidation of the structure of the remodeled nucleosome. (2) Determination of the roles of histone modifications in remodeling. (3) Elucidation of the role of the Isw1p remodeling complex. (4) Comparison with chromosomal HIS3. Our work on CUP1 and HIS3 indicates that, at least for these two genes, the target of remodeling complexes is a domain rather than just the promoter. This is an important finding, because it suggests that remodeling complexes act on chromatin domains. What is the function of domain remodeling? We speculate that remodeling entire genes might facilitate elongation through nucleosomes by RNA polymerase II. In a wider context, the fact that remodeling complexes can participate in the formation of chromatin domains might be important in understanding the formation of domains in higher eukaryotes. RNA Polymerase II Phosphorylation and Transcription through Nucleosomes This project is part of a continuation of our previous work aimed at deciphering the mechanism of transcription through the nucleosome. Although RNA polymerase II is able to transcribe through a nucleosome in vitro, it is exceedingly slow, far slower than the measured rates of transcription through chromatin in vivo. This has led many investigators to try to identify factors which might dramatically improve the transcription rate through the nucleosome in vitro. One possibility is a role for the phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. CTD phosphorylation occurs in vivo as RNA polymerase II is converted from its initiating form to the elongation complex. It is therefore the phosphorylated form of the polymerase that is responsible for transcription through nucleosomes in vivo. In this work, we compared the rates of transcription through the nucleosome by phosphorylated and unphosphorylated polymerase. However, CTD phosphorylation had little effect on the rate. Thus, if CTD phosphorylation has a role in transcription through the nucleosome in vivo, it must be indirect.